Abstract
The human gastrointestinal tract harbors a diverse and complex microbiome, which interacts in a variety of ways with the host. There is compelling evidence that gut microbial dysbiosis, defined as an alteration of diversity and abundance in intestinal microbes, is an etiological factor in inflammatory bowel disease (IBD). Membrane vesicles (MVs), which are nano-sized particles released by bacteria, have been found to interact with the host and modulate the development and function of the immune system. As a result MVs have been suggested to play a critical role in both health and disease. In this study we developed a method to isolate, characterize and assess the immunoreactivity of heterogeneous populations of MVs from fecal samples (fMVs) of healthy volunteers. We successfully isolated 2*109-2*1010 particles/ml from 0.5 gram of feces by using a combination of ultrafiltration and size exclusion chromatography (SEC) from 10 fecal samples. Bead-based flowcytometry in combination with tunable resistive pulse sensing (TRPS) provided a reliable method for (semi-)quantitative determination of fMVs originating from both Gram-positive and Gram-negative bacteria, while transmission electron microscopy confirmed the presence of fMVs. Real time 16s PCR on bacterial cell fractions or isolated fMVs DNA of the most common phyla (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria) revealed differences in the relative abundance between bacteria and the fMVs. Moreover, fMVs evoke the release of TNF- by THP-1 cells in a dose-dependent matter. Also, a significant positive correlation was found between Actinobacteria/-Proteobacteria derived vesicles and the release of TNF-. It has become increasingly clear that fMVs could provide an additional layer to the definition of homeostasis or dysbiosis of the microbiota. The current study supports their potential involvement in the intestinal homeostasis or inflammatory disorders and provides putative interesting incentives for future research.
Highlights
The human gastrointestinal tract harbors a diverse and complex microbiome, which interacts in a variety of ways with the host
To confirm that these samples predominantly contained vesicles and not proteins, a Bradford assay was performed in combination with tunable resistive pulse sensing (TRPS), see Supplementary Figures S1A and B
As we are predominantly interested in the bacteria-derived membrane vesicles in feces, we optimized the bead-based technique which we have previously developed to measure extracellular vesicles
Summary
The human gastrointestinal tract harbors a diverse and complex microbiome, which interacts in a variety of ways with the host. Evidence is accumulating suggesting additional associations between microbial dysbiosis and a variety of diseases affecting the pulmonary and cardiovascular systems (Dang and Marsland, 2019; Tang et al, 2019) as well as the liver (Minemura and Shimizu, 2015) and even the brain (Foster and McVey Neufeld, 2013). Molecular mechanisms underlying these effects have been elucidated to some extent. More recently it has been suggested that membrane vesicles, naturally released by most types of bacteria, may contribute to both health and disease and as such be important mediators by which the microbiome affects the host (Cecil et al, 2019; Macia et al, 2019)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
